Flow cytometry is used conveniently to measure quickly one or more optical parameters of the cells and/or particles that pass through a light beam that impinges on a narrow examination zone. Background information on flow cytometry is found, for example, in Shapiro's Practical Flow Cytometry, Third Edition (Alan R. Liss, Inc. 1995), incorporated herein by reference in its entirety.
In conventional flow cytometers, sample fluid containing sample cells and/or particles is introduced from a sample tube into or near the center of a faster flowing stream of sheath fluid, which draws the sample toward the center of the combined streams. This process, known a hydrodynamic focusing, allows the cells and/or particles to be delivered reproducibly to the center of the measuring point in the flow cell or other examination zone. Typically, the cells and/or particles are in suspension in the flow cell.
A continuous wave laser focuses a laser beam on the cells and/or particles as they pass through the examination zone in a flow stream of the suspension. When an object of interest in the flow stream is struck by the laser beam, certain signals result and are sensed by detectors. For example, these signals include forward light scatter intensity, which provides information concerning the size of individual cells and/or particles, and side light scatter intensity, which provides information regarding the relative size and refractive properties of individual cells and/or particles.
Other signals which may be sensed by detectors include fluorescence emissions from one or more fluorescent dyes and/or other fluorescent molecules; for example, tryptophan, or other fluorescent amino acid(s) or other molecule(s), that is native to a protein or other peptide, or to another biomolecule or man-made molecule. Typically, when a plurality of different fluorescing molecules is employed in an analytical scheme, fluorescence emission peaks are selected so as to minimize or, ideally, eliminate spectral overlap between the respective fluorescence emission peaks.
For example, ideally, fluorescence emission peaks will differ by 50 nm, though lesser (or larger) spectral separation also can be accommodated and used to advantage, for example, 20, 30 or 40 nm, where the greater the spectral separation, the more powerful is the discrimination between the respective fluorescence emitters. In addition, quantum efficiency is considered in the choice of fluorescent molecules. In the case of use of a plurality of different fluorescent molecules, a separate detector is tuned to and used for the different wavelength of emission of each fluorescence emitter. The excitation wavelength for more than one fluorescent molecule can be the same, or different excitation wavelengths can be used to match the excitation spectrum of each different fluorescent molecule.
The optical signals that are generated by an analytical procedure are transmitted to an output meter(s) and/or data storage means. Signal processing, for example, by a Digital Signal Processor or DSP, can be employed before and/or after intensity data on the measured optical signals are transmitted to the output meter(s) and/or data storage means.
In flow cytometry, an “event” occurs when a cell or particle passes through the beam of the laser or other light source. As the event progresses, the light measured from scattering and/or fluorescence emission increases as the cell or particle enters the beam, reaches a maximum at the center of the beam, and tapers to a nominal value as it leaves the beam.
State of the art flow cytometers commonly use one of two systems to measure events. One system uses peak detectors, including peak hold circuits, to sustain the maximum signal level obtained from an event, and to measure optical parameters relating to cells and/or particles passing through a laser beam. Once a peak detector has sensed and measured an event, the peak detectors are turned off to provide sufficient time for an analog to digital, or A/D, converter to digitize that maximum value of the signal. During the time that the peak detectors are off, termed “dead time”, any event occurring within the laser beam will go undetected.
The second system uses an integrator to measure the area under the peak collected for an event. With either system, when an event occurs within the cytometer's light beam during the dead time, the event will go undetected.
Normally, such lack of detection of an event does not constitute a significant problem because, for a large number of events, only a tiny number will be missed. However, applications which require the detection of rare, critical events, for example, identification of one special cell or particle in a thousand or a million, suffer from the possible non-detection of a rare event when current generation flow cytometers are used for detection and measurement. The higher the throughput, the greater is the probability that such a critical event will be missed per unit time period.
One solution to problems encountered in sorting samples in flow cytometry is provided by U.S. Pat. No. 5,550,058 to Corio, et al., incorporated herein by reference. Corio, et al. provides a means of flexibly controlling decisions on the sorting of events detected in a flow cytometer. Events are pre-qualified according to user-selectable parameters to permit reduction of events missed during dead time. However, the Corio, et al reference does not teach or suggest a means of reducing the probability of missing a dead time event to zero.
Thus, it would be beneficial in the art of flow analyzers, including flow cytometers or other analyzing equipment, to have a means to reduce to zero the probability of missing any event, including a rare, dead time event.
It is also desirable to provide efficiently and inexpensively a system and/or method for reducing the probability of missing an event.
It is further desirable to provide efficiently and inexpensively a system and/or method for reducing the probability of missing an event for high sampling rates, for example, on the order of approximately a sampling period, such as one sample per millisecond or shorter period.